Ligation of Synthetic Peptides to Proteins Using Semisynthetic Protein trans-Splicing

Abstract

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

An Atypical Mechanism of Split Intein Molecular Recognition and Folding

“…Beyond the mechanistic implications of atypical split intein association, the Cat intein presented herein should be a powerful tool for protein semisynthesis. Cat is the fastest and most robust atypical split intein reported to date, and it should therefore find immediate utility in the N-terminal modification of expressed proteins. ,, This functionality should complement other reported methods for protein N-terminal modification, such as expressed protein ligation, transpeptidase-based ligation strategies, artificially split inteins, and various protein chemistry methods. − Furthermore, under specific contexts, the extein preferences observed will favor the application of Cat in protein engineering: Cat exhibits promiscuous activity in the presence of substitutions within the C-extein but is highly vulnerable to mutations within the N-extein. This preference is complementary to the commonly applied naturally split DnaE inteins, which are sensitive to C-extein mutations. , …”

“…This site is proximal to the C-terminus, generating N- and C-inteins of approximately 100 and 35 amino acids, respectively (Figure b). However, split inteins with atypical split sites have recently been reported from metagenomic sequencing data and characterized in vitro (Figure b). , These atypically split inteins contain N-inteins of ∼30 amino acids, making them accessible to solid-phase peptide synthesis and suitable tools for the N-terminal modification of recombinant proteins. , Like canonical split inteins, atypically split inteins must be capable of molecular recognition through a specific and efficient association event that results in the formation of a folded HINT domain. Although the HINT domain displays pseudo 2-fold symmetry, the respective C- and N- fragments of canonically and atypically split inteins do not mirror one another in a structural sense (Figure c).…”

Split Inteins

“…The hydrophobicity of the lipid modification requires the use of liposomes and nicely underlines the range of conditions under which these natively split inteins are active. The synthetic intein-extein pieces can also be used to deliver other cargos like synthetic fluorophores for protein labeling, as described previously in this book series [11] and also in Chapter 8. Here, Julio A. Camarero and Radhika Borra even take synthetic protein labeling by protein trans-splicing to proteins in living mammalian cells.…”